Development of an LC-MS/MS method for determining 5-MeO-DIPT in dried urine spots and application to forensic cases.

PURPOSE: The dried urine spots (DUSs) technique is increasing continuously as an easy sampling method for monitoring substance abuse due to its advantages of stability and convenience regarding transport and storage. 5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is a new type of tryptamine hallucinogen, the use of which has been banned in many countries. And according to the previous research, 5-MeO-DIPT is not stable in urine. In order to improve its stability, an LC-MS/MS method for determining 5-MeO-DIPT in DUSs was developed. METHOD: 10 µl urine was spotted on Whatman FTATM classic card, then extracted with 200 µl methanol, and liquid chromatography-tandem mass spectrometry in positive ion multiple reaction monitoring mode was utilized for analysis. RESULTS: The LOD and LLOQ of the method were 0.1 ng/ml and 0.2 ng/ml, respectively. The accuracy and precision were 98.2%-103.9% and 2.7%-8.5%, respectively. It was found that the stability of 5-MeO-DIPT in DUSs was better than the stability of 5-MeO-DIPT in urine stored at 25 °C. Moreover, this method was also applied to detect 5-MeO-DIPT in the urine of individuals known to have used 5-MeO-DIPT. It was found that the concentrations of 5-MeO-DIPT were 0.3-2.3 ng/ml, which were lower than those obtained via GC-Orbitrap-MS. The small volume of urine required (10 µl), combined with the simplicity of the analytical technique, makes this an useful procedure for the screening of drug of abuse.

Determination of 5-MeO-DIPT in Human Urine Using Gas Chromatography Coupled with High-Resolution Orbitrap Mass Spectrometry.

5-Methoxy-N,N-Diisopropyltryptamine (5-MeO-DIPT) is a designer hallucinogen derived from tryptamine and its use has been banned by many countries. In this study, a qualitative and quantitative method was developed for determining 5-MeO-DIPT in urine by gas chromatography high-resolution mass spectrometry. 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT) and 5-methoxy-N-isopropyltryptamine (5-MeO-IPT) were identified as 5-MeO-DIPT metabolites in abusers' urine. 5-MeO-DIPT was extracted from urine by liquid-liquid extraction with ethyl acetate under alkaline conditions. The extract was analyzed by GC-Orbitrap-MS in full scan mode with a resolution of 60,000 full width at half maxima (FWHM). The linear range of this method was 2-300 ng/mL with r > 0.99, and the limit of detection was 1 ng/mL. The accuracy and precision were 93-108.7% and 3.1-10.3%, respectively. This method is simple and sensitive. It has been successfully used to detect 5-MeO-DIPT in drug abusers' urine, which showed that the concentrations of 5-MeO-DIPT were between 1 and 2.8 ng/mL. 5-OH-DIPT and 5-MeO-IPT, two urinary major metabolites of 5-MeO-DIPT, were identified in urine samples from 5-MeO-DIPT users. Furthermore, the stability of 5-MeO-DIPT in human urine was investigated. It was discovered that the concentration of 5-MeO-DIPT in urine decreased by 22.8, 33.2 and 38.2% after samples were stored for 24 h at 25°C, 5 days at 4°C and 7 days at 4°C, respectively. And 5-MeO-DIPT in urine were stable after they were stored for 30 days at -20°C. Therefore, it is recommended that urine should be stored under freezing conditions before performing 5-MeO-DIPT analysis.

Metabolism of the tryptamine-derived new psychoactive substances 5-MeO-2-Me-DALT, 5-MeO-2-Me-ALCHT, and 5-MeO-2-Me-DIPT and their detectability in urine studied by GC-MS, LC-MSn , and LC-HR-MS/MS.

Many N,N-dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5-methoxy-2-methyl-N,N-diallyltryptamine (5-MeO-2-Me-DALT), 5-methoxy-2-methyl-N-allyl-N-cyclohexyltryptamine (5-MeO-2-Me-ALCHT), and 5-methoxy-2-methyl-N,N-diisopropyltryptamine (5-MeO-2-Me-DIPT) using gas chromatography-mass spectrometry (GC-MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC-MSn ), and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC-HR-MS/MS. 5-MeO-2-Me-DALT (24 phase I and 12 phase II metabolites), 5-MeO-2-Me-ALCHT (24 phase I and 14 phase II metabolites), and 5-MeO-2-Me-DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O-demethylation, hydroxylation, N-dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O-demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N-dealkylation. For SUSAs, GC-MS, LC-MSn , and LC-HR-MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC-MS SUSA, both LC-MS SUSAs were able to detect an intake of 5-MeO-2-Me-ALCHT and 5-MeO-2-Me-DIPT via their metabolites following 1 mg/kg BW administrations and 5-MeO-2-Me-DALT following 0.1 mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd.

A quantitative method for simultaneous determination of 5-methoxy-N,N-diisopropyltryptamine and its metabolites in urine using liquid chromatography-electrospray ionization-tandem mass spectrometry.

5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is a designer hallucinogen derived from tryptamine and is reportedly abused and involved in criminal activities. For the detection of 5-MeO-DIPT use, a liquid chromatography-tandem mass spectrometric method for 5-MeO-DIPT and its metabolites, 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT) and 5-methoxy-N,N-isopropyltryptamine (5-MeO-IPT) was developed and validated in rat urine. The urine samples were pretreated by protein precipitation with acetonitrile and introduced into a BDS HYPERSIL C(18) column (50 × 2.0 mm, 5 µm) for chromatographic separation. Mobile phases consisted of methanol, water, and 1% formic acid, and gradient elution was used at a flow rate of 0.2 mL/min. For the MS detection, multiple-reaction monitoring analysis was adopted. The linear range was 0.01-10 µg/mL, and the lower limit of quantification was 10 ng/mL for all analytes. The intra- and interday accuracies and precisions met the criteria (<15%). The developed method was successfully applied to the drug-treated rat urine.

Acute confusional state after designer tryptamine abuse.

A 23-year-old Japanese woman was brought to the emergency department about 6.5 h after taking liquid and later a half tablet purchased on the street. About 4.5 h prior to presentation, she displayed excited and disorganized behavior. On examination, she was not alert or oriented, with a Glasgow Coma Scale score of 13, did not answer any questions from doctors while smirking and looking around restlessly, and sometimes exhibited echolalia, imitating the speech of doctors. She was given intravenous infusion of fluid for 8 h, then discharged. Gas chromatography-mass spectrometry of urine revealed 5-methoxy-diisopropyltryptamine, 5-methoxy-N-methyltryptamine and an unidentified tryptamine. Identifying chemical products based solely on information of users is insufficient, and urinalysis is necessary in cases potentially involving designer drugs.

A fatal poisoning with 5-methoxy-N,N-diisopropyltryptamine, Foxy.

A fatal overdose involving case by 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is reported. 5-MeO-DIPT and its two metabolites, 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT) and 5-methoxy-N-isopropyltryptamine (5-MeO-NIPT), were identified by LC-MS. The level of 5-MeO-DIPT, 5-OH-DIPT and 5-MeO-NIPT in blood and urine was 0.412, 0.327 and 0.020 microg/ml, and 1.67, 27.0 and 0.32 microg/ml, respectively. These blood and urine levels were higher than published data for such poisoning.

Metabolism of the psychotomimetic tryptamine derivative 5-methoxy-N,N-diisopropyltryptamine in humans: identification and quantification of its urinary metabolites.

The urinary metabolites of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) in humans have been investigated by analyzing urine specimens from its users. For the unequivocal identification and accurate quantification of its major metabolites, careful analyses were conducted by gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, and liquid chromatography-tandem mass spectrometry, using authentic standards of each metabolite synthesized. Three major metabolic pathways were revealed as follows: 1) side chain degradation by O-demethylation to form 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT), which would be partly conjugated to its sulfate and glucuronide; 2) direct hydroxylation on position 6 of the aromatic ring of 5-MeO-DIPT, and/or methylation of the hydroxyl group on position 5 after hydroxylation on position 6 of the aromatic ring of 5-OH-DIPT, to produce 6-hydroxy-5-methoxy-N,N-diisopropyltryptamine (6-OH-5-MeO-DIPT), followed by conjugation to its sulfate and glucuronide; and 3) side chain degradation by N-deisopropylation, to the corresponding secondary amine 5-methoxy-N-isopropyltryptamine (5-MeO-NIPT). Of these metabolites, which retain structural characteristics of the parent drug, 5-OH-DIPT and 6-OH-5-MeO-DIPT were found to be more abundant than 5-MeO-NIPT. Although the parent drug 5-MeO-DIPT was detectable even 35 h after dosing, no trace of its N-oxide was detected in any of the specimens examined.

Sensitive determination of alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine using gas chromatography-mass spectrometry.

We devised a sensitive and simple method to determine alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine, using gas chromatography-mass spectrometry (GC-MS). AMT and 5MeO-DIPT were extracted using an Extrelut column with an internal standard, bupivacaine, followed by derivatization with acetic anhydride. The derivatized extract was used for GC-MS analysis of EI-SIM mode. The calibration curves of AMT and 5MeO-DIPT were linear in the concentration range from 10 to 750 ng/ml in both blood and urine samples. The method detection limit (MDL) of AMT and 5MeO-DIPT were 1 ng/ml each in whole blood and 5 ng/ml each in urine. This method should be most useful to accurately determine the presence of these drugs in blood and urine in clinical and forensic cases.

A foxy intoxication.

Foxy is the colloquial name for the hallucinogen 5-ethoxy-diisopropyltryptamine (5-MeO-DIPT). A non-fatality involving a 23-year-old Caucasian man who ingested a capsule containing 5-MeO-DIPT is described. He presented to the Emergency Department, not with visual nor auditory hallucinations but with sensory hallucinations, that of formication and paranoia. He was observed and given supportive care for 4 h, then discharged without any known sequelae. Blood and urine were collected for laboratory analyses. Foxy and its metabolites were identified in urine by gas chromatography-mass spectrometry. The concentrations of 5-MeO-DIPT in the serum and urine were 0.14 and 1.6 microg/mL, respectively. The drug undergoes oxidative deamination to form 5-methoxy-indole acetic acid. The urinary concentration of this metabolite was 0.17 microg/mL. Also, the urine contained three other related compounds. Two of them have been described in a previous case of 5-MeO-DIPT ingestion as 5-methoxy-isopropyltryptamine (5-MeO-IPT) and 5-methoxy-diisopropyltryptamine-N'-oxide (5-MeO-DIPT-N'-oxide). The third compound was substantially present in the urine and was tentatively identified as 5-hydroxy-diisopropyltryptamine (5-OH-DIPT). Only the parent drug, 5-MeO-DIPT was detected in the serum sample.

A general screening and confirmation approach to the analysis of designer tryptamines and phenethylamines in blood and urine using GC-EI-MS and HPLC-electrospray-MS.

Recent additions of designer tryptamines and phenethylamines to the Drug Enforcement Administration's schedule of controlled substances necessitate analytical procedures for their detection and quantitation. As specific immunoassays are not currently available and cross-reactivities with existing assays are unknown, a screening method based on gas chromatography-mass spectrometry was developed. The method was capable of measuring the pentafluoropropionic derivatives of a-methyltryptamine (AMT), N,N-dimethyltryptamine (DMT), 4-bromo-2,5-dimethoxy-beta-phenethylamine (2CB), N,N-dipropyltryptamine (DPT), 2,5-dimethyl-4-N-propylthio-beta-phenethylamine (2C-T-7), and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DiPT). Separation was optimized to allow tentative identification of metabolites, which display common electron impact ionization fragmentation patterns. The screening method gave limits of detection between 5 and 10 ng/mL and demonstrated linearity between 50 and 1000 ng/mL. The method was successfully applied to blood and urine samples in suspected AMT intoxications. Confirmation of 5-MeO-DiPT in one of the subjects' urine was achieved using liquid chromatography-mass spectrometry (LC-MS). Quantitation by selected ion monitoring (SIM) yielded a urinary concentration of 229 ng/mL. The method was linear from 25 to 1500 ng/mL with a correlation coefficient of 0.995. The limit of detection was 5 ng/mL in urine on the LC-MS. Two additional peaks were observed and presumed to be metabolic products reported previously as 5-methoxy-N-isopropyltryptamine (5-MeO-iPT) and 5-methoxy-N,N-diisopropyltryptamine-N'-oxide (5-MeO-DiPT-N-oxide).

Foxy, a designer tryptamine hallucinogen.

Foxy is slang for 5-methoxy-N,N-diisopropyltryptamine. It has hallucinogenic properties, similar to other tryptamine compounds, and is mildly euphoric. This case report describes a 21-year-old Caucasian man who ingested a pill called Foxy containing an unknown amount of drug. He was observed in hospital for 2 h, during which time he had mild hallucinations and could not move his limbs. A urine sample was collected approximately 4 h after drug ingestion. The patient was then discharged with no follow up assessment. The 5-methoxy-N,N-diisopropyltryptamine was identified in the urine by gas chromatography-mass spectrometry. Standards prepared from the pure material were used in the identification. Quantitative analysis using the same analytical technique resulted in a urinary concentration of 1.7 micro g/mL. Through oxidative deamination, the metabolite, 5-methoxy-indole acetic acid, was formed. It was identified in the urine, and the concentration was determined to be 1.3 micro g/mL using gas chromatography-mass spectrometry. Two other compounds were discovered in the urine sample as a result of a routine drug screen. From their mass spectra, they were tentatively identified as 5-methoxy-N-isopropyltryptamine and 5-methoxy-N,N-diisopropyltryptamine-N'-oxide.